Abstract: A gateway-compatible bimolecular fluorescence complementation（BiFC） assay system as well as a low-cost vector for entry cloning was constructed. With newly designed entry vector and gateway technology， target genes were quickly recombined to the BiFC destination vectors and introduced to tobacco leaves with Agrobacterium-mediated injection， and the interactive fluorescence under confocol microscope was observed. Gateway entry vector developed in this study has a very low background for gene cloning. Due to the ccdB gene， the nonlinear vector cannot survive in ccdB sensitive strains after transformation， thus eliminating the background of the vector. After the digestion of XcmⅠ， the linearized entry vector has a T overhang at 3’， and the PCR product can be efficiently ligated to the vector as same as a commercial T vector. Therefore the vector developed in this study can be easily prepared in laboratory and applied to TA cloning， which greatly reduced the cost for entry cloning. With the BiFC destination vectors produced in this study， the target gene can be quickly recombined to the destination vectors and used for gene interaction assay. With the gateway system developed in this study， genes can be efficiently cloned and applied to interaction assay with a lower cost.

Key words: entry vector, BiFC, gene interaction, Gateway